The role of salinity in recovery of white sturgeon (Acipenser transmontanus) from stimulated angling stress

White sturgeon (Acipenser transmontanus) may be acclimated to different salinities in the Fraser River, which may impact their ability to recover from severe exercise induced by angling. Sturgeon acclimated to different salinities and subjected to stimulated angling stress showed differences in recovery rate of physiological parameters.


Introduction
White sturgeon (WS) Acipenser transmontanus (Richardson, 1863) is the largest freshwater fish species in North America. They are believed to have existed as a species for >46 million y demonstrating their resilience to major geological and climatic changes over this time (Shedko, 2022). Unfortunately, due to human activities associated with overharvesting, pollution, dam construction and habitat destruction over the past 150 y, they are now endangered or threatened throughout much of their historical range (Hildebrand et al., 2016). In British Columbia, the lower and middle Fraser River contains a population of WS of approximately 44 000 fish (Nelson et al., 2019), which supports a year-round catch and release (C&R) fishery that draws considerable tourism revenue (Glova et al., 2009). Although WS are the dominant sturgeon species in the Fraser River, green sturgeon (A. medirostris) are known to enter the lower Fraser River infrequently from the Salish Sea but are not a target for the C&R fisheries, and reports of capture in fresh water (FW) are rare (COSEWIC, 2004). Despite WS being heavily managed, there is long-term decline with changes to population structure that include fewer younger fish due to limited recruitment (Nelson et al., 2013(Nelson et al., , 2019. Primarily due to conservation concerns of this iconic fish, there have been numerous studies investigating the impact of C&R on WS populations (McLean et al., 2016(McLean et al., , 2019(McLean et al., , 2020. Experimental work suggests that WS recover quickly from C&R angling in general and seldom experience mortality (McLean et al., 2016(McLean et al., , 2019(McLean et al., , 2020, with an estimated direct mortality of angled fish <0.012% and 3-d postangling mortality rate of 2.6% (Robichaud et al., 2006). Angling events can vary, with fight times ranging from seconds to >2 h with a median time of 5.78 min (McLean et al., 2016), and WS are likely captured multiple times over their life. During capture events, WS often spend time out of water; however, they are tolerant of air exposure provided their gills are kept moist, as Shartau et al. (2017) found WS could recover from a 45-min air exposure.
The physiological challenges associated with C&R are largely due to stress related to exhaustive exercise during angling capture as fish struggle (Wood et al., 1983). Exhaustive exercise in WS occurs while fish attempt to avoid capture, inducing a severe metabolic acid-base disturbance whereby large reductions in blood and red blood cell (RBC) pH are observed, along with smaller but significant reductions in tissue pH (Shartau et al., 2017). This response is a welldocumented pattern in fishes (Randall and Brauner, 1991;Nelson et al., 1996;Harter et al., 2014). The tolerance of WS to the severe acid-base disturbance associated with exercise likely accounts for the limited mortality observed during C&R because severe acidification of white muscle is implicated as a likely cause of death in fish (Wood et al., 1983). The effect of exercise on sturgeon is altered by various factors including water temperature, exercise duration and intensity, as well as fish age, sex and life-stage (McLean et al., 2016(McLean et al., , 2020, making experimental exploration challenging. Fishing for WS on the lower Fraser River occurs in both the nontidal freshwater (FW) region and the higher salinity tidal region, which extends >20 km upstream; in fact, the tidal effect can reach as far upstream as 84 km (Murray, 2016;Leung et al., 2018). This salt wedge creates a stable region of higher salinity due to the higher density of salt water and may result in WS being acclimated to salinities ranging from FW up to 20 (parts per thousand), which occurs at the mouth of the river. Although WS can tolerate salinity (McEnroe and Cech, 1985;Vaz et al., 2015), there may be physiological differences in the response during, and after, C&R fishing due to the physiological differences of fish acclimated to different salinities. Differences in salinity can elicit changes in the physiological response of fishes (Byrne et al., 1972;Redding and Schreck, 1983;Wood, 1991;Nelson et al., 1996;Pedersen and Malte, 2004;Allen et al., 2014;Christensen et al., 2018), including those related to acid-base disturbances (Randall and Brauner, 1991), likely a result of the osmotic and ionic differences incurred by fish between FW and seawater Damsgaard et al., 2020).
Bouts of exhaustive exercise, such as that arising from C&R i) increase blood and tissue lactate due to increased anaerobic metabolism, ii) induce a rapid reduction in blood pH (pH e ) (referred to as a metabolic acidosis) and iii) changes plasma ions and hematocrit, which are associated with water imbalance and loss of ion homeostasis. In some species, a reduction in tissue pH accompanies the metabolic acidosis (Ferguson and Tufts, 1992;Pankhurst and Dedualj, 1994;Nelson et al., 1996). Recovery from acid-base disturbances typically occurs over the following 8-48 h, although water composition may alter the severity and recovery profiles (Larsen and Jensen, 1997;Tovey and Brauner, 2017;Sackville et al., 2018). Consequently, the capacity to recover from severe C&R-induced exercise may differ depending on water salinity. Both an increase in severity and a decrease in recovery rate of the associated acidosis could induce exercisedinduced mortality. As in trout, mortality has been linked to the large acid load incurred during exhaustive exercise and their inability to successfully compensate for it through typical pH regulatory mechanisms (Wood et al., 1983).
Because WS from the lower Fraser River may be acclimated to different salinities, they may also experience different physiological responses as a result of C&R; these differences may translate into more severe outcomes for both short-term and long-term survival. In addition, because exhaustive exercise is a highly stressful event (Baker et al., 2005b;McLean et al., 2016), there are elevations in stress indicators, such as plasma cortisol and glucose, which are known to have long-term effects on whole animal function (Faught et al., 2016) and can have maternal effects in progeny (Faught and Vijayan, 2018). Consequently, C&R capture of gravid females under differing salinities, for example, could exacerbate reproductive effects and negatively impact recruitment in the Fraser River in unforeseen ways.
Although sturgeon demonstrate the capacity to tolerate exhaustive exercise in FW (Kieffer et al., 2001;Baker et al., 2005a;Shartau et al., 2017;Brown and Kieffer, 2019;Penny and Kieffer, 2019;Kieffer and May, 2020), it remains unknown how quickly they are able to restore their acid-base status and it is uncertain if acclimation to higher salinities affects severity of, or recovery from, this challenge. To examine the effect of salinity on exhaustive exercise associated with C&R, we subjected WS to stimulated angling  stress (SAS) after acclimation to either 0, 10 or 20 and assessed the effects using a suite of physiological indicators. These three salinity levels were chosen because they represent FW, estuarine and dilute seawater found in the Salish Sea near the mouth of the Fraser River (Murray, 2016;Leung et al., 2018). These hyposaline, isosaline and hypersaline waters, respective to the fish, have significantly different driving forces for both ions and water movement. To further characterize the role of salinity on SAS, we assessed recovery from this challenge at both 4 and 8 h postexercise, within which time partial or complete recovery would generally be expected (e.g. Baker et al., 2005b). This work was designed to illustrate whether environmental salinity (and presumably anadromy) may play an important role not only in the exercise-based behaviours of fish, such as predator avoidance, prey capture or migration, but also to provide insight into whether C&R for WS in saline waters elevates the risk of mortality.

Animal acquisition and holding
All experiments were performed at the International Center for Sturgeon Studies (ICSS) at Vancouver Island University (VIU). WS were reared from hatch at ICSS from wild captive brood stock from a single male:female cross, and maintained in large, indoor flow-through tanks (23 m 3 ) in dechlorinated City of Nanaimo tap water [61 μmol l −1 Na + , 69 μmol l −1 Cl − (City of Nanaimo, 2015), pH ∼6.6-6.8 (Amiri et al., 2009)] at ∼15 • C under a simulated natural photoperiod and were fed daily to satiation using a 24-h belt feeder based on aquaculture produced feeding tables (Conte et al., 1988). WS (n = 78) used in this study were 3 y old and had a mean weight of 2.97 ± 0.06 kg. Food was withheld 48 h before manual chasing and experimental sampling. Experiment protocols were approved by the Vancouver Island University Animal Care Committee (Animal Usage Protocol: 2018-04-R).

Salinity acclimation
WS were divided into three experimental groups (n = 26 per group) of different salinities (0, 10 and 20 ) in stand-alone tanks (1.8 × 1.8 × 0.7 m) with a total density per tank of approximately 33.5 kg m −3 . The 0 treatment group was reared in dechlorinated City of Nanaimo tap water (as previously), whereas fish in the other two groups were created by adding calculated amounts of Instant Ocean ® dissolved in that same water, increasing salinity 3-4 every 2 d until the desired salinity was reached. Aeration provided via an air compressor was provided within each tank to maintain dissolved oxygen at >80% of air saturation. Water changes of 25-50% were done as needed (roughly every 2 d) to keep ammonia levels below acceptable thresholds (∼0.01 mg/L) (Meade, 1985). Sturgeon were fed 0.5% body weight daily (EWOS brood feed for salmonids-4 and 5 mm, 50% protein). Fish were acclimated to higher salinities for at least 2 wk before experimentation and showed no evidence of ill health associated with salinity acclimation. Experiments with all three salinities were run simultaneously (i.e. held at identical times in identical tanks at similar densities), with all sampling done through 4 d from 18-22 December 2018 to avoid seasonal effects.

Stimulated angling stress and recovery
Sturgeon were removed from tanks and subjected to a twostep exhaustive exercise protocol previously used (Shartau et al., 2017) to stimulate angling stress. Fish were chased with a plastic stick for 5 min or until the fish was completely exhausted, allowed to rest for 5 min, then chased again until complete exhaustion (i.e. no further escape response exhibited to being chased). Fish were then placed in individual aerated recovery tanks (1 × 0.5 × 0.5 m) matching the salinity of that of each group and allowed to recover for 0.5, 4 or 8 h. Control fish were sampled directly from the respective holding tanks.

Blood sampling, tissue sampling and ions
After the appropriate recovery period, fish were rapidly removed from the recovery box and killed by cephalic concussion, after which blood (8 mL) was drawn caudally via a syringe (10-mL syringe, 23 G 3.1-cm needle) and placed on ice. After this procedure, complete hearts were removed within 2 min, gently squeezed and patted dry to remove blood, placed in aluminum foil and immediately placed in liquid N 2 and then transferred to an ultracold freezer (−80 • C) at the end of the day. Blood was divided into two aliquots. Blood pH, Hct, lactate and glucose were measured from one aliquot; the other aliquot was centrifuged for 3 min at 10 000 rpm and plasma was removed for measurement of total CO 2 (TCO 2 ), osmolarity and [Cl − ].
Blood pH was measured using a glass electrode (Radiometer Analytical SAS pH electrode; GK2401C, Cedex, France or Orion 8302BNUMD ROSS Ultra pH/ATC Triode) connected to a pH meter (Orion Star A211 pH meter, ThermoFisher Scientific, Waltham, MA, USA). Intracellular pH (pH i ) of heart tissue and RBC were measured using the same glass pH electrode previously described. RBC pH i was measured using the freeze-thaw method as described and validated previously (Zeidler and Kim, 1977;Baker et al., 2009). Tissue pH i was measured using the metabolic inhibitor tissue homogenate method (MITH) and the pH electrode previously mentioned (Pörtner et al., 1991;Baker et al., 2009). Plasma TCO 2 was measured using a TCO 2 analyser (Corning model 965 Analyzer); the remaining plasma was used to measure [Cl − ] ions (HBI model 4 425 000; digital chloridometer), osmolarity (model 5520; Westcor Vapor Pressure Osmometer), lactate (Lactate Pro™) and glucose (Onetouch™ Ultra2) (Baker et al., 2005b).

Plasma [HCO 3
− ] and PCO 2 were calculated using TCO 2 and pH values as described by Brauner et al. (2004). solubility coefficient and the logarithmic acid dissociation constant (pK') for plasma were determined based on previous work by Boutilier et al. (1984). All values are expressed as mean ± SEM throughout; n = 8 for all treatments except where otherwise noted. Data were compared by Welch t test or, where multiple treatments were evaluated, data were analysed by an analysis of variance (ANOVA), followed by Tukey post hoc test to compare all groups with each other. When two-way ANOVA interactive terms were significant, the factors were analysed separately using a one-way ANOVA. When data did not meet normality (Shapiro-Wilk normality test) or equal variance (Bartlett test) assumptions, a Kruskal-Wallis test followed by Dunn multiple comparison test was used (p < 0.05) to confirm conclusions. GraphPad Prism (v.8) was used for all statistical analyses and for preparation of figures.

Results
As evident from the lack of differences amongst plasma osmolarity measurements from different salinities (Fig. 1A), WS exhibited complete acclimation to 10 and 20 over the time period allowed. Neither salinity exposure nor exhaustive exercise to simulate the stress of angling imposed by manual chasing resulted in mortality in any treatment group of animals.

Osmoregulatory changes
Exposure to higher salinities did not significantly alter plasma osmolarity, although, as is typical in many marine fishes when compared with FW fishes (Edwards and Marshall, 2013), osmolarity of fish in 0 tended to be lower at 261 mOsM compared with 273 and 272 mOsM in 10 and 20 , respectively (Fig. 1A). Overall, plasma osmolarity was signifi-cantly different among salinity (F 2,64 = 9.49, P < 0.001), time (F 3,64 = 19.68, P < 0.001), but not the interaction (Table 1), with a post hoc test revealing that plasma osmolarity was significantly higher in individuals at 0.5 h than control fish; osmolarity was no longer different than control fish after 8 h (Fig. 3C).

Extracellular acid-base status
SAS resulted in changes to blood acid-base in all salinity groups ( Fig. 2A). Blood pH (pH e ) was reduced in all treatment groups 0.5 h postexercise. The mean pH e across all salinities was reduced from 7.70 ± 0.02 to 7.44 ± 0.02 at 0.5 h, with pH e values remaining depressed between 0.5 and 4 h postexercise, but almost identical to control values by 8 h in all three groups ( Fig. 2A). Differences in pH e occurred in the time after SAS (F 3,64 = 54.04, P < 0.001) ( Table 1); there was no effect of salinity on pH e (F 2,64 = 2.817, P = 0.067), with the exception of 0.5 h postexercise between the 10 and the 20 groups revealed by a post hoc test. The pattern of changes in blood PCO 2 were similar to those of pH e , because values were elevated at 0.5 h postexercise in all salinity groups, returning to control values by 8 h (Fig. 3A). Kruskal-Wallis test revealed PCO 2 changes after SAS were significant in all three salinities (0 -H = 15.35 (2), P < 0.01; 10 -H = 14.85 (2), P < 0.01; 20 -H = 15.73 (2), P < 0.01), with post hoc test uncovering differences in PCO 2 between 0.5 and 8 h at all three salinities. Differences in PCO 2 between salinities at each time point occurred 4 (F 2,16 = 16.37, P < 0.001) and 8 h (F 2,17 = 12.19, P < 0.001) after SAS, with post hoc test indicating differences between 0, 10 and 20 (Fig. 3A). SASinduced changes in plasma [HCO 3 − ] with both interval postexercise (F 3,62 = 9.75, P < 0.0001) and salinity (F 2,62 = 17.54, P < 0.0001) having significant effects (Fig. 3B). A post hoc test indicated that 4 h postexercise, plasma [HCO 3 − ] was significantly lower in the 10 and 20 groups both compared with the time-matched 0 group and the preceding 0.5 h postexercise group. Similarly, the post hoc test indicated that at 8 h, fish acclimated to 0 had [HCO 3 − ] significantly elevated over that of fish acclimated to 10 (Fig. 3B).

Intracellular pH
Salinity acclimation did not result in significant changes to RBC pH (F 2,12 = 2.523, P = 0.12; Fig. 2B) (Table 1)  differences were present at 0.5 h post-SAS (F 2,16 = 14.65, P < 0.001), with a post hoc test indicating the 10 and 20 groups were not different from each other, but were both lower than the 0 group. RBC pH largely recovered by 8 h post-SAS but significant differences in pH remained (F 2,16 = 4.29, P < 0.05); post hoc test indicated this was due to differences between the 0 and 20 groups. There was a significant effect of time postexercise (F 3,62 = 3.10, P < 0.05) and salinity acclimation (F 2,62 = 3.81, P < 0.05) on heart pH, with post hoc analysis indicating that heart pH i in the 10 group was significantly lower than those in the FW group at 0.5 h post-SAS (Fig. 2C).

Secondary stress indicators
Salinity treatment did not affect blood glucose concentrations, either before or after the angling challenge. Changes in blood glucose did occur after SAS at each salinity level (0 -H = 17.62 (2), P < 0.001; 10 -F 3,21 = 6.288, P < 0.01; 20 -F 3,21 = 7.385, P < 0.01) and resulted in roughly a doubling of glucose levels at 0.5 h postexercise, with post hoc analysis indicating a significant increase in blood glucose at 4 and 8 h in all salinities compared with controls (Fig. 4A).

Discussion
Three-year old white sturgeon acclimated to 10 and 20 WS aged 3 y were able to acclimate to elevated salinity of 10 or 20 through a 2-wk period. No significant differences were observed in plasma osmolarity among groups after salinity treatment (Fig. 1A), and these values were similar to those reported by other studies measuring osmolarity in WS (McEnroe and Cech, 1985;Tashjian et al., 2007;Amiri et al., 2009;Shaughnessy et al., 2015), although less than those of juvenile Siberian sturgeon (A. baerii), Chinese sturgeon (A. sinensis), and Persian sturgeon (A. persicus) acclimated to 14 (Rodríguez et al., 2002), 10 (He et al., 2008) or 11 (Shirangi et al., 2016), respectively. The small but insignificant increase in plasma osmolarity between FW and 10 and 20 is likely due in part to the change in plasma [Cl − ], which increased from 104.5 mM in FW to and 113.4 and 120.4 mM in 10 and 20 , respectively (Fig. 1B). Despite these increased levels, values for osmolarity and plasma [Cl − ] in WS were slightly lower than the values for a typical euryhaline teleost (Edwards and Marshall, 2013); this has been previously noted (Shaughnessy et al., 2015).
As an anadromous species, WS spawn and spend the first part of their life in FW, and at some point, they become capable of entering seawater. Unlike salmonids, another group of anadromous fishes, smoltification in sturgeon is not characterized by known morphological, behavioural and physiological changes; however, changes in hormones (e.g. growth hormone) and Na + , K + -ATPase do occur (Hasegawa et al., 1987;Rodríguez et al., 2002;Allen et al., 2011). Anadromous sturgeon, including WS, will enter seawater after a couple of years of FW residence (Zydlewski and Wilkie, 2013); the exact timing of seawater entry, although not clear, is believed to be dictated by a combination of age and size. Evidence for this is in the finding that larger juvenile WS experience lower mortality when transferred to elevated salinity (Zydlewski and Wilkie, 2013). In addition, McEnroe and Cech (1985) found that in the 0.4-to 0.6-g size class, there was only 50% survival after transfer to 10 and <5% survival after transfer to 15 ; not until fish reached 4.9-9.5 g did survival reach 100% after transfer to both salinities; similar findings were observed by others (Amiri et al., 2009) and also in gulf sturgeon (A. oxyrhynchus desotoi) (Altinok et al., 1998). Interestingly, WS do not seem to have a "smoltification window" beyond which salinity acclimation is not possible, as occurs in some salmonids (pink and chum salmon) (Hasegawa et al., 1987).
Salinity acclimation, as indicated by seawater entry, varies amongst anadromous species. Together, the aforementioned findings indicate that although sturgeon do have some capacity for salinity tolerance at early life stages, they are not capable of surviving long-term in hyperosmotic full-strength seawater at less than 30 g (Amiri et al., 2009). Previous exposure to brackish water does seem to make the transition from FW to seawater (SW) less challenging though (McKenzie et al., 2001). The closely related green sturgeon spend long portions of their life history in marine waters and brackish estuaries (Lindley et al., 2008), and unsurprisingly seem capable of earlier seawater entry than other sturgeon species because they reached 100% survival to 20 at 60 d posthatch (<5 grams) and 100% survival to 30 and 34 at 100 and 135 d posthatch, respectively (Allen and Cech, 2007;Lindley et al., 2008;Hildebrand et al., 2016). Sea lamprey (Petromyzon marinus) have an FW phase (ammocoete) that typically lasts for 3-7 y followed by the parasitic phase at sea (McCormick, 2013;Zydlewski and Wilkie, 2013). Another basal fish species, alligator gar (Atractosteus spatula), can survive as a juvenile (<1 y, 185 g) in salinities <24 for >30 d, but longer term survival depends on returning to lower salinities (Schwarz and Allen, 2014). Differences exist between the closely related Pacific salmonids, where pink (Oncorhynchus gorbuscha) and chum (O. keta) enter seawater soon after hatch, whereas others such as coho (O. kisutch) and steelhead trout (O. mykiss) may spend one to several years in FW (McCormick, 2013). WS, in contrast to those aforementioned examples, tend to remain in FW or brackish environments, with limited marine excursions (Hildebrand et al., 2016). This migratory pattern of returning to FW to recover from saline excursions may be similar for small WS, and may also explain why

Metabolic acidosis after exercise
Salinity acclimation did not alter resting blood pH, nor did the severity of acid-base disturbance in the blood after SAS differ among salinity treatments. Blood pH was reduced by an average of 0.26 pH units 0.5 h after SAS ( Fig. 2A). Exerciseinduced metabolic acidosis is a common response observed amongst fishes (Wood, 1991;Milligan, 1996;Nelson et al., 1996;Harter et al., 2014), including WS (Shartau et al., 2017). The changes in pH e reported here are similar to those reported by Shartau et al. (2017), who observed a reduction in pH e from 7.7 to 7.4 0.25 h post-SAS in WS in FW.
RBC pH i after acclimation to 20 was significantly lower than either FW or 10 (P < 0.05). At 0.5 h after SAS, RBC pH i was reduced in 10 , 4 h in 0 (P < 0.05), which recovered by 8 h. In the 20 group, RBC pH i did not significantly change; this may be a result of the lower initial starting pH i . RBC pH i in resting FW fish in this study was 7.56 pH units, which was higher than the previously reported value of 7.2 pH units by Shartau et al. (2017) and 7.35 pH units by Baker et al. (2009). Interestingly, Shartau et al. (2017) observed a reduction in RBC pH i 0.25 h postexercise in FW sturgeon exercised to exhaustion using a similar methodology-here, no significant reduction was observed. This odd difference suggests that either the pH i reduction was not captured by the time points sampled here (and may be compensated for exceedingly quickly) or the severity of the exercise was perhaps less.
Heart pH i remained unchanged after SAS at all salinities; however, there were insignificant reductions occurring within the first 0.5 h (Fig. 2C), which is similar to what was observed by Shartau et al. (2017), who reported a small but significant reduction in heart pH 2 h postexercise in 0 fish. The reduction seems to be the most pronounced in the 10 , and although it is not statistically significant, this may represent a change in capacity for heart pH i regulation. Salinity acclimation is associated with altered gene expression: gill tissue of Siberian sturgeon (Guo et al., 2018), medaka (Oryzias melastigma) (Liang et al., 2021), stickleback (Gasterosteus aculeatus) (Gibbons et al., 2017), and Argyrosomus japonicus (Li et al., 2021) all exhibited differential expression of genes associated with ion transport and cell permeability to ions and water. Medaka also exhibited changes in differential expression of genes in liver associated with protein synthesis and metabolism (Liang et al., 2021). Given the changes observed in other species, it is likely WS also experience changes in gene and protein expression after salinity. This exposure may alter how tissues, such as the heart, regulate ions, including those associated with pH regulation, resulting in biologically relevant impacts on heart function given that small pH changes may have consequences for transporter function and activity (Parks et al., 2008). Of particular interest for further study may be that, in WS, an isoosmotic environment may induce expression of genes that are substantially different than those induced by a hyperosmotic one. The timing of the exposure could very well also play an important role in this migratory fish.

Impact of salinity acclimation on recovery from SAS
Acclimation to salinities similar to those found in the Fraser River and Fraser River estuary did not alter the capacity of 3y-old WS to recover from SAS because the severity of acidosis and time course of recovery were similar between salinity treatments. Even so, differences in plasma [Cl − ], [HCO 3 − ], [PCO 2 ], and lactate concentrations suggest that salinity acclimation may alter the mechanisms involved in acidbase homeostasis associated with exercise-induced metabolic acidosis, which may have consequences for regulation of tissue pH.
Changes in recovery pattern between salinities (Fig. 5) indicate that at 4 h postexercise, fish in 10 and 20 exhibited a metabolic acidosis, whereas fish in 0 did not. Because only sufficient numbers of sturgeon were available to examine four time points, this may not have allowed full capture of all changes, as previously, Shartau et al. (2017) found that WS in FW experienced a metabolic acidosis within 0.25 h postexercise, and this persisted for at least 2 h. That there was no metabolic acidosis apparent in FW fish in this study suggests that either recovery happened more rapidly in the current study or that the severity of the exercise was less than in Shartau et al. (2017).
The modest differences between the responses of WS to SAS in differing salinities are likely associated with changes to transporter isoform expression and activity in tissues during salinity change. In particular, acclimation to increased salinity induces significant changes in transporter isoforms and activity at the gills. In other sturgeon species, transfer from FW to brackish water induces an increase in Na+-K+-ATPase (NKA) activity (Rodríguez et al., 2002;He et al., 2008;Shirangi et al., 2016); this was also observed in other euryhaline fishes (Richards et al., 2003;Scott et al., 2004;Evans et al., 2005;Nilsen et al., 2007;Urbina et al., 2013;Esbaugh et al., 2019). These changes in NKA activity and expression can also occur in the kidneys (Marshall and Grosell, 2005;Yang et al., 2016) and intestines (Fuentes et al., 1997;Schwarz and Allen, 2014). In green sturgeon acclimated to high salinity, upregulation of NKCC (Na+, K+, 2Cl− transporter) and downregulation of V-type H + -ATPase (VHA) occurred in the gills (Sardella and Kültz, 2009). In Atlantic salmon gills, changes to NKCC and cystic fibrosis transmembrane conductance regulator (CFTR) have been observed after acclimation to higher salinity (Nilsen et al., 2007). Certainly, the possibility that similar changes in WS could drive differences in the capacity or timing of the organismal response to SAS is worth examining, especially in light of potential mortality of an endangered species impacted by a C&R fishery. Many fishes including WS are able to preferentially regulate pH i , which provides tremendous capacity for pH i regulation during acid-base disturbances; however, this strategy has only been examined in FW-acclimated fish (Shartau et al., 2016(Shartau et al., , 2019(Shartau et al., , 2020. The changes in ion transporters at the tissue level described previously may subsequently affect pH i regulation, which in sturgeon is likely one of the most protective responses to an acid-base disturbance. The capacity for pH i regulation of RBC and heart seems to be reduced in higher salinities, which may suggest changes to transporter isoforms and activity when responding to higher salinities has consequences for pH i regulation. Regulation of pH i has been examined after SAS and fishes using preferential pH i regulation seem to be largely capable of protecting pH i after exercise (Harter et al., 2014;Shartau et al., 2017), although this may represent a greater challenge than respiratory acidosis in WS, perhaps due to high lactate concentration (this study; Shartau et al., 2017). However, should pH i regulation be more difficult in WS acclimated to higher salinities, the potential increase in recovery time may expose large WS to greater predation or higher incidental mortality. Thus, whether mechanisms of preferential pH i regulation yet to be described are less effective at high salinity is a topic that should be investigated.

Concluding points
Acclimation within 2 w of 3-y-old WS to salinities up to 20 does not pose a challenge, and once acclimation is achieved, it does not seem to greatly change their response to SAS.
Because WS support economically important C&R fisheries in British Columbia (Glova et al., 2009), knowing how they recover from SAS informs on their ability to recover from C&R (McLean et al., 2016(McLean et al., , 2019(McLean et al., , 2020. Due to the salt wedge that extends up the Fraser River (Murray, 2016;Leung et al., 2018), WS may be acclimated to different salinities within the region where C&R fisheries occurs. Based on these results, there are not likely any physiological challenges facing WS after the SAS if they are caught in the different salinity regions of the Fraser River. In addition, because sturgeon of the size and age examined here were capable of acclimation, future studies aimed at determining when between the size of 30 g (Amiri et al., 2009) and 3-y-old, 3-kg WS (this study) become salinity tolerant would provide further insight into the migratory patterns of young WS. It may be important for conservation managers and researchers to consider how other environmental factors alter recovery from SAS. This may include changes in temperature and dissolved oxygen because these are known to impact fish recovery from exercise (Randall and Brauner, 1991;Deslauriers and Kieffer, 2012;Mandal et al., 2016), and upstream regions of the Fraser River are likely to experience greater variation in these environmental parameters compared with those further downstream nearest the ocean.
Even though the risk of C&R mortality seems to be unaffected by salinity acclimation, a further risk might be associated with the saline waters in the Fraser. Should an FW-or SW-acclimated fish be caught and then returned into water of different salinity, risk factors for mortality may be higher, particularly if recovery is slower than normal. As this study suggests, salinity acclimation alters the pattern by which recovery from the metabolic acidosis occurs, it warrants further investigation of various scenarios that could

Conflicts of Interest Statement
The authors have no conflicts to declare.

Data Availability
Datasets are available on request: The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.